Field of the Invention
The invention relates generally to labeled protein standards for use in biochemical separations and more specifically to labeled protein standards for used in gel electrophoresis.
Background Information
Tools that aid in the development of new drugs and new medical diagnostics, as well as certain diagnostics themselves, require accurate and efficient analysis of protein samples. This in turn requires markers that accurately allow the identification of the size of proteins in a protein sample that is separated using separation methods. Separation methods that are commonly performed in biochemistry for the purification, identification, and characterization of proteins include chromatography, gel electrophoresis, and solution electrophoresis. These methods typically use standards for molecular weight or charge determination. Gel electrophoresis in particular is a common tool for the development of new drugs and medical diagnostics that is typically performed with molecular weight markers.
Pre-labeled protein standards for electrophoresis are notoriously less sharply resolving than unlabeled standards, and often the molecular weights of the labeled markers are inexact, differing from the unlabeled proteins by varying amounts. The bands of a pre-stained protein marker run in a denaturing polyacrylamide gel can be, for example, significantly wider and more diffuse than a band that results from the same protein that has not been pre-labeled, but instead is stained after electrophoresis is complete. This is largely due to the difficulties in uniformly labeling a particular protein standard.
Labeling of proteins is typically performed by attaching a label to a chemical group of one or more amino acid residues of the protein. The significant reactive groups of amino acids behave as nucleophiles in chemical reactions, for example, the sulfhydryl group of cysteine; the amino group of an N-terminal amino acid or of lysine, histidine, tryptophan, or arginine; the carboxyl group of aspartate and glutamate or a C-terminal amino acid; the phenolate of tyrosine; and the thioether of methionine. The selection of a particular reactive chemical group on the dye to be conjugated to a protein and manipulation of reaction conditions at which a chemical conjugation is performed (such as, for example, pH) will typically favor conjugation of a dye to one or more particular amino acids.
Although reaction conditions can be adjusted to reduce side reactions with one or more amino acids that are not targeted for labeling, side reactions are difficult to completely eliminate or control. The addition of label to a variable number of sites of a particular protein through side reactions reduces the uniformity in the amount of label attached to the protein, such that a given labeled protein standard comprises a population of labeled protein molecules in which different members of the population have different migration characteristics. Pre-labeled standards therefore typically do not resolve as well as unlabeled proteins in separations, producing bands on electrophoresis gels, for example, that are much less sharp than the bands produced by the same proteins electrophoresed in unlabeled form. The variability of labeling of pre-labeled standards often makes molecular weight determination using pre-labeled standards unreliable.
Another factor contributing to poor resolution of pre-labeled proteins on electrophoresis gels is protein-to-protein variability in the ratio of the number of attached dye molecules to molecular weight. Because a protein standard set uses different marker proteins to represent different molecular weights, and the different proteins of the set have variable ratios of the number of target amino acid residues to molecular weight, it is often necessary to mix different amounts of individual labeled protein standards to provide a pre-labeled marker set having proteins with similar intensity for visualization of the marker proteins. In many cases, this requires that one or more labeled proteins will be “overloaded” in a gel lane with respect to protein amount to achieve a desirable intensity for the resulting band on an electrophoresis gel. The overloading of proteins of the standard set leads to bands on the gel that are broad and not sharply delineated, making it difficult to assess the migration distance of the protein of a particular molecular weight.